Voltage-gated calcium channels are a heterogeneous family of membrane proteins, which respond to depolarization by opening a calcium-selective pore through the plasma membrane. The influx of calcium into cells mediates a wide variety of cellular and physiological responses including excitation-contraction coupling, hormone secretion and gene expression. In neurons, calcium entry directly affects membrane potential and contributes to electrical properties such as excitability, repetitive firing patterns and pacemaker activity. Calcium entry further affects neuronal function by directly regulating calcium-dependent ion channels and modulating the activity of enzymes such as protein kinase C and calcium-dependent endent calmodulin-dependent protein kinase II. An increase in calcium concentration at the presynaptic nerve terminal triggers the release of neurotransmitters. Calcium entry also plays a role in neurite outgrowth and growth cone migration in developing neurons and has been implicated in long-term changes in neuronal activity.
In addition to the variety of normal physiological functions mediated by calcium channels, they are also implicated in a number of human disorders. Recently, mutations identified in human and mouse calcium channel genes have been found to account for several disorders including, familial hemiplegic migraine, episodic ataxia type 2, cerebellar ataxia, absence epilepsy and seizures. (Fletcher, C. F., et al., Cell (1996) 87:607–617; Burgess, D. L., et al., Cell (1997) 88:385–392; Ophoff, R. A., et al., Cell (1996) 87:543–552; Zhuchenko, O., et al., Nature Genetics (1997) 15:62–69. The clinical treatment of some disorders has been aided by the development of therapeutic calcium channel modulators or blockers. (Janis, R. J., et al., Calcium Channels: Their Properties, Functions, Regulation and Clinical Relevance (1991) CRC Press, London).
Native calcium channels have been classified by their electrophysiological and pharmacological properties as either high voltage-activated (L, N, P, and Q types) or low voltage-activated channels (T-type). R-type channels have biophysical properties similar to both high and low voltage-activated channels. (For reviews see McCleskey, E. W., et al., Curr. Topics Membr. (1991) 39:295–326, and Dunlap, K., et al., Trends Neurosci. (1995) 18:89–98.) T-type channels are a broad class of molecules that transiently activate at negative potentials and are highly sensitive to changes in resting potential. The L, N, P and Q-type channels activate at more positive potentials and display diverse kinetics and voltage-dependent properties. There is some overlap in biophysical properties among the high voltage-activated channels, consequently pharmacological profiles are useful to further distinguish them. L-type channels are sensitive to dihydropyridine (DHP) blockers, N-type channels are blocked by the Conus geographus peptide toxin, ω-conotoxin GVIA, and P-type channels are blocked by the peptide ω-agatoxin IVA from the venom of the funnel web spider, Agelenopsis aperta. A fourth type of high voltage-activated Ca2+ channel (Q-type) has been described, although whether the Q- and P-type channels are distinct molecular entities is controversial (Sather, W. A., et al., Neuron (1993) 11:291–303; Stea, A., et al., PNAS (1994) 91:10576–10580), and it has been suggested that they result from alternative splicing of a single gene (Bourinet, et al., “Phenotypic variants of P- and Q-type calcium channels are generated by alternative splicing of the α1A subunit gene.” Nature Neuroscience (1999) 2:407–415. Conductance measurements of several types of calcium channels have not always fallen neatly into any of the above classes and there is variability of properties even within a class, suggesting that additional calcium channels subtypes remain to be classified.
Biochemical analyses show that neuronal calcium channels are heterooligomeric complexes consisting of three distinct subunits (α1, α2δ and β) (reviewed by De Waard, M., et al., Ion Channels, Volume 4, (1997) edited by Narahashi, T., Plenum Press, New York). The α1 subunit is the major pore-forming subunit and contains the voltage sensor and binding sites for calcium channel blockers. The mainly extracellular α2 is disulphide-linked to the transmembrane δ subunit. Both are derived from the same gene and are proteolytically cleaved in vivo. The β subunit is a non-glycosylated, hydrophilic protein with a high affinity of binding to a cytoplasmic region of the α1 subunit. A fourth subunit, γ, is unique to L-type Ca channels expressed in skeletal muscle T-tubules. The isolation and characterization of γ-subunit-encoding cDNA's is described in U.S. Pat. No. 5,386,025, which is incorporated herein by reference.
Molecular cloning has revealed the DNA sequence and corresponding amino acid sequences of seven different types of α1 subunits (α1A, α1B, α1C, α1D, α1E, α1F and α1S) and four types of β subunits (β1, β2, β3 and β4) (reviewed in Stea, A., et al., Handbook of Receptors and Channels (1994) Edited by R. A. North, CRC Press). PCT Patent Publication WO 95/04144, which is incorporated herein by reference, discloses the sequence and expression of α1E calcium channel subunits. More recently, several α1 subunits corresponding to the low voltage gated T-type calcium ion channel have been cloned. Descriptions of these cloned α1 subunits may be found, for example, in PCT publications WO 98/38301 and WO 01/02561 as well as in U.S. Pat. Nos. 6,309,858 and 6,358,706, all incorporated herein by reference.
The different classes of α1 and β subunits have been identified in a variety of mammals including rat, rabbit and human, and share a significant degree of amino acid conservation across species—for example see:                For β: Castellano, A., et al., J. Biol. Chem. (1993) 268:3450–3455;                    Castellano, A., et al., J. Biol. Chem. (1993) 268:12359–12366;            Perez-Reyes, E., et al., J. Biol. Chem. (1992) 267:1792–1797;            Pragnell, M., et al., FEBS Lett. (1991) 291:253–258;                        For α1: Dubel, S. J., et al., Proc. Natl. Acad. Sci. USA (1992) 89:5058–5062;                    Fujita, Y., et al., Neuron (1993) 10:585–598;            Mikami, A., et al., Nature (1989) 340:230–233;            Mori, Y., et al., Nature (1991) 350:398–402;            Snutch, T. P., et al., Neuron (1991) 7:45–57;            Williams, M. E., et al., Science (1992) 257:389–395;                        Both α & β: Soong, T. W., et al., Science (1993) 260:1133–1136;                    Tomlinson, W. J., et al., Neuropharmacology (1993) 32:1117–1126;            Williams, M. E., et al., Neuron (1992) 8:71–84.                        
In some expression systems the α1 subunits alone can form functional calcium channels although their electrophysiological and pharmacological properties can be differentially modulated by coexpression with any of the four β subunits. Until recently, the reported modulatory affects of β subunit coexpression were to mainly alter kinetic and voltage-dependent properties. More recently, it has been shown that β subunits also play crucial roles in modulating channel activity by protein kinase A, protein kinase C and direct G-protein interaction. (Bourinet, E., et al., EMBO J. (1994)13:5032–5039; Stea, A., etal., Neuron (1995) 15:929–940; Bourinet, E., etal., Proc. Natl. Acad Sci. (USA) (1996) 93:1486–1491.)
Genes have been identified that encode four different but homologous α2δ subunits. The first subunit identified was α2δ-1 in rabbit skeletal muscle. Five tissue-specific splice variants exist (Angelotti, T., et al., FEBS Lett. (1996)397:331–337). α2δ-2, -3 and -4 have been identified recently in human and mouse (Klugbauer, N., et al., J. Neuroscience (1999) 19(2):684–691; Qin, N., et al., Mol. Pharmacol. (2002) 62(3):485–496). These α2δ subunits share 30% to 56% amino acid sequence identity with the α2δ-1 subunit as well as several structural motifs, such as similar hydrophobicity profiles, glycosylation sites and cysteine residues. α2δ-1 and α2δ-2 subunits are expressed in many tissues including the brain and heart, while α2δ-3 is only found in the brain. α2δ-4 is distributed in certain cell types of the pituitary, adrenal gland, colon and fetal liver. α2δ-2 has been proposed as a tumor suppressor gene, and the mouse homolog is a candidate for the ducky epileptic phenotype (Gao, B., et al., J. Biol. Chem. (2000) 275(16): 12237–12242).
In general, the α2δ subunit does not function alone as a calcium ion channel, but rather is used in combination with the α1 subunit and optionally β, and in the case of L-type subunits, optionally a γ subunit.
The α2δ-1 subunit increases the current density of calcium channels by increasing the amount of functional channel at the cell surface and enhances dihydropyridine binding to L-type channels and ω-conotoxin GVIA to N-type channels (Brust, P. F., et al., Neuropharmacology (1993) 32(11):1089–1102; Felix, R., et al., J. Neurosci. (1997) 17(18):6884–6891). α2δ-2 and α2δ-3 significantly enhance and modulate the current through a number of HVA and LVA channels (Hobom, M., et al., Eur. J. Neurosci. (2000) 12(4):1217–1226). Gabapentin, an antiepileptic, has been shown to bind to α2δ-1 and α2δ-2 but not to α2δ-2 but not to α2δ-3 (Marais, E., et al., Molec. Pharmacol. (2001) 59(5):1243–1248).